Material layer volume determination

ABSTRACT

A technique for generally determining the volume of a layer of a constituent material in a centrifugally separated mixture of material. A centrifuge tube is used to hold the material mixture and an appropriately shaped body is disposed in the tube in the zone occupied by the constituent material whose volume is to be measured. The body reduces the available volume within the tube which may be occupied by the constituent material, and thus expands the axial extent of the constituent material to make visual measurement of the constituent material more accurate.

This invention relates to a technique for enabling quick visualmeasurement of the approximate volume of a constituent material layer ina centrifuged material mixture. More particularly, this inventionemploys a body disposed in a centrifuge tube within the general confinesof the layer to be measured and which acts to elongate the axial extentof the volume occupied by the layer to be measured.

Various techniques have been proposed for use in measuring the volume ofa constituent layer of material within a material mixture, whichmaterial mixture has been centrifuged to separate out the variousconstituent materials into layers according to density, or specificgravity. These techniques have found particular application in measuringthe several constituents of various biological fluids, such as blood,for example.

The particular blood constituent which has proven to be most difficultto quickly and easily measure is the buffy layer, which is made up ofthe various white cell types and platelets. In a centrifugedanticoagulated whole blood sample, the buffy layer is located betweenthe red cell layer and the plasma layer, but because of the relativepaucity of the buffy layer, a quick visual technique for measuring thebuffy layer has, heretofore, been lacking.

One technique for determining the white cell count involves the use of aprecisely measured volume of whole blood which is precisely diluted andplaced in an optical counting chamber of a given volume. The dilutedblood sample is then examined with a microscope and the leukocytes orwhite blood cells are visually counted. This technique is timeconsuming, requires relatively expensive equipment, and is subject toerrors arising from inaccurate sample measurement and imprecise sampledilution.

Another technique has been devised for the automatic measurement of theleukocyte count of a blood sample. The sample of whole blood is manuallyor automatically diluted and the white cells are counted either bydetecting and measuring light scatter from them in the sample as theypass through a confined space, or by measuring their effect on anelectrical field as they pass through a small aperture. These automatedtechniques are quite accurate, but the equipment needed is quiteexpensive. The equipment also requires specially trained technicians tobe used.

In the more general field of the volumetric measurement of constituentmaterial layers in a centrifuged material mixture, it has been proposedto physically enlarge the axial extent of the layer of interest in orderto perform some further operation on the layer. Specifically, it isproposed in the prior art to provide a specially formed flask for use indetermining the leukocyte count in a whole blood sample. The flask is acentrifuge vessel and includes a mid-axial area of constricted internaldiameter which, because of its reduced volume, will form a narrowvertical column of leukocytes. A high density material, such as mercury,must be used in this flask to raise the blood sample so as to ensurethat the leukocytes occupy the narrow mid-axial neck of the vessel. Onceproperly positioned, the leukocytes are aspirated out of the vessel andsubjected to further testing. It will be appreciated that such a flaskis difficult to accurately form, is quite fragile, and cannot be readilyused as a capillary tube due to the necessity of having one end closedto facilitate the introduction of the mercury thereinto. This flask thusis used for relatively large blood samples and the harvesting ofleukocytes therefrom only.

Another proposal for centrifugal separation enhancement is set forth inthe prior art. This latter technique involves the use of closely packedartificially produced microspheres which are diposed within a centrifugetube to restrict the free volume available to be occupied within thetube by the centrifuged fluid material. The microspheres are formed witha plastic core having a metallic coating for accurately controlling theoverall sphere density. Sphere densities, in turn, are selected so thata plurality of spheres will float in each layer of the centrifugedmaterials. Thus each material layer will have its particular densityequal to the density of an associated plurality of spheres, whichspheres, by reason of the density gradiant of the centrifuged mixture,will be restricted to and "float in" the particular material layer ofthe same density. In this manner the axial extent of each layer will beexpanded for easier visual measurement. The spheres of differentdensities can also be of different colors to further contrast theseveral layers each from the others. This technique has several problemsone of which relates to centrifugation in general, i.e. the problem ofhow to get the spheres into the tube, especially if the tube is ofcapillary size, and the other of which relates to its use in a buffylayer measuring technique. This latter problem arises from the packingcharacteristics of spheres wherein the resultant free space available tobe occupied by the buffy layer is about 33% of the original free spacepresent without the spheres. This means that the axial extent of thebuffy layer in a centrifuged blood sample can only be increased aboutthreefold using microspheres alone, and this degree of increase isinsufficient to permit simple, visual measurement of the volume of thebuffy layer with any acceptable degree of accuracy.

In order to provide a quick, inexpensive visual technique fordetermining the general buffy layer measurement or leukocyte count in ananticoagulated blood sample, we propose the use of at least one axiallyelongated volume-occupying mass which is made of a material having aspecific gravity such that the mass will float upon, or slightly in thered cell layer of the centrifuged blood sample. The mass is disposed inthe capillary tube bore, has its axis of elongation substantiallycoincidental with the tube bore axis and combines with the tube bore toform an axially extending free space between the tube bore wall and theexterior of the mass. The volume of the free space is substantially lessthan the free volume of the tube bore so that the axial extent of thebuffy layer will be markedly increased when the buffer layer ispositioned in the free space during and after centrifugation.

The geometric form of the mass can vary widely, as will be outlined ingreater detail hereinafter. The mass can also be used in combinationwith a microsphere cluster for further enhancement. Furthermore, themass can be used for visual identification of the several internalconstituents of the buffy layer, as will be outlined hereinafter.

It is, therefore, an object of this invention to provide a technique foruse in visually determining the general leukocyte and platelet countwhich make up the buffy layer in a sample of whole blood.

It is a further object of this invention to provide a technique of thecharacter described which entails the use of a capillary-type centrifugetube containing an axially elongated volume-occupying mass selectivelypositioned in the buffy layer and floating on the red cell layer of theblood sample.

It is yet another object of this invention to provide a technique of thecharacter described which utilizes expendable paraphenalia and can beperformed quickly in a doctor's office by relatively untrainedpersonnel.

It is yet another object of this invention to provide a technique of thecharacter described for performing a differential white cell andplatelet count.

It is an additional object of this invention to provide a technique ofthe character described which is relatively accurate, and inexpensive touse.

These and other objects and advantages of the invention will become morereadily apparent from the following detailed description of severalembodiments of the invention taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is an exploded perspective view of one embodiment of an apparatususable in determining the leukocyte or white cell and platelet count inaccordance with the invention;

FIG. 2 is an axial sectional view of a capillary tube employed inaccordance with prior art teachings for centrifugation of whole bloodsample into its constituent components, i.e. red cells, white cells,platelets and plasma;

FIG. 3 is an axial sectional view of the apparatus of FIG. 1 as it isemployed to make a visual measurement of the white cell and plateletcount in a centrifuged whole blood sample;

FIGS. 4-6 are perspective views of several variations of the insertportion of the apparatus of FIG. 1;

FIG. 7 is an axial sectional view of the modified insert of FIG. 5 takenalong line 7--7 thereof;

FIG. 8 is an axial sectional view of the modified insert of FIG. 6;

FIG. 9 is an axial sectional view of a specimen tube showing anembodiment of the invention which includes interphase-demarcatingmicrospheres of particulate material of appropriate specific gravitiesused in conjunction with an axially elongated volume-occupying body ofthe type shown in FIG. 1,

FIGS. 10 and 11 are perspective views of further embodiments of aninsert which may be used as the volume-occupying mass in this invention.

Referring now to the drawings, there is shown in FIG. 1 one embodimentof an apparatus which can be used in accordance with the invention formaking a visual determination of the approximate white cell and plateletcount in a sample of whole blood. The apparatus includes a capillarytube 2 of conventional construction having a through bore 4 which isopen at both ends of the tube 2. An axially elongated volume-occupyingmass 6 is positioned inside of the tube bore 4. In the embodiment shownin FIG. 1, the mass 6 takes the form of a right cylindrical insert orplug which is composed of a material having a predetermined specificgravity which renders the mass buoyant upon the centrifuged red cellmass. Due to its shape, the insert 6 will be held in the tube bore 4 sothat both have substantially coincidental axes at all times. Thediameter of the insert 6 is sufficiently smaller than the diameter ofthe tube bore 4 so as to be slidable within the tube bore 4 so that theinsert 6 can gravitate to the red cell layer during centrifugation ofthe blood sample and float upon the red cell layer after centrifugation.

The difference between the respective diameters of the insert 6 and tubebore 4 will form a free space of restricted volume, which free space isoccupied by the centrifuged layer of white cells and platelets. Byrestricting the size of the free space available to the buffy layer, theapparent height or thickness of the buffy layer will be expanded overthat obtained in an unrestricted capillary tube bore. Changes in thevolume of the buffy layer from sample to sample will thus be "magnified"so that one can visually determine whether the white cell and plateletcount is high, low, or average, generally speaking. This generaldetermination will then be used to indicate whether further moresophisticated tests are needed. It will be appreciated that the degreeof expansion of the height of the buffy layer can be varied by varyingthe difference between the diameters of the insert 6 and tube bore 4.Expansion factors in the range of four up to twenty may be obtained. Forexample, an expansion factor or multiple of nine is readily obtained. Itwill be appreciated that this apparent expansion of the buffy layer isthe result of the fact that the insert 6 occupies volume within the tubeadjacent to the red cell layer, and thus reduces the free volumeavailable to the buffy layer by a multiple of 0.75 or more.

FIG. 2 illustrates a capillary tube 4 containing a blood sample whichhas been centrifuged down to its constituent component layers. It willbe noted that the lower end of the tube bore has been sealed by a dab ofclay, wax, or the like 3 prior to centrifugation. The red cell layer isdenoted generally by the letter R, the white cell and platelet layere.g. the buffy layer, by the letter B, and the plasma layer by theletter P. It will be noted that the axial extent or thickness of thewhite cell and platelet layer B is diminimus thus making it impossibleto visually determine generally whether the white cell and plateletcount is abnormally high, low, or average.

Referring now to FIG. 3, there is shown the apparatus of FIG. 1 used toaxially elongate the bounds of the buffy layer B. The sample has beencentrifuged in the tube 2 with the volume-occupying mass insert 6 inplace within the tube bore 4. As can be noted from FIG. 3 and aspreviously described, the tube bore 4 and the side surface of the insert6 combine to form an annular free volume V directly above the red celllayer R into which annular free volume the buffy layer settles duringcentrifugation. It will also be noted that the annular free volume V issubstantially smaller than a corresponding free volume within the tubebore, and thus the axial extent of the buffy layer occupying the annularfree volume is substantially expanded. In other words, the distancebetween the upper and lower menisci of the buffy layer is increased overthat shown in FIG. 2. A minimum expansion multiple of about four isconsidered to be useful in visual white cell and platelet countdetermination in accordance with this invention. If so desired, areference guide 8 may be used for comparison with the apparatus fordetermining high, low, or average white blood cell and platelet count.The guide 8 may display reference indicia 10 for alignment with theupper and lower menisci of the buffy layer. Thus a measurement of thedistance between the upper and lower menisci of the buffy layer is madeto determine the white cell and platelet counts. More precisemeasurements of the relative axial expansion of the buffy layer by theuse of mechanical, optical or electrical means can also be made from theapparatus of FIG. 3.

The apparatus of FIGS. 1 and 3 is used as follows. The insert 6 ispositioned inside of the tube bore 4 and the ends of the tube 2 may beinwardly crimped as at 1 to retain the insert within the bore, or theinsert may be adhered to the wall of the tube bore by means of ablood-soluble adhesive such as gum acacia. The tube is then used in aconventional manner to draw a blood sample from a patient by means of afinger prick or the like. The drawn sample is then centrifuged with theresultant separation and elongation of the buffy layer as shown in FIG.3.

It will be noted that the axially elongated shape of the insert a lendsitself to formation thereof by extrusion of a synthetic resinous meltand subsequent cutting of the extrudate to length. The insert may alsobe made by injection molding of a resinous melt. The insert 6 is made ofa material or materials having a specific gravity or cross-sectionalbulk density in the range of 1.02 gm/cc to 1.09 gm/cc, and preferablyabout 1.04 gm/cc so that the insert 6 will be buoyant upon the red celllayer and yet sink through the buffy layer. Examples of such a materialare acrylonitrile butadiene styrene (ABS), "commercial" styrene, and MMAstyrene copolymer. A layering of different density materials may also beused to form the insert so long as the cross-sectional bulk density ofthe layered insert is an appropriate value.

The shape of the insert 6 shown in FIGS. 1 and 3 is right cylindrical,however FIGS. 4, 5, 6 and 10 disclose other insert configurations whichcan be used without departing from the spirit of the invention.

FIG. 4 discloses an insert 12 which is provided with one or more axialchannels 14 formed in its side surface. The channels 14 form passages inwhich the buffy layer will settle during centrifugation.

FIG. 5 discloses an insert 16 which has a cylindrical side wall 18. Anaxial channel 20 into which the buffy layer settles is formed in theside wall. The channel 20 has a restricted mouth 22 at its lower endwhich is adjacent to the red cell layer, and the volume of the channel20 expands at a logarithmic rate from the mouth 22 to its upper end 24.The use of a logarithmic or other non-linear rate of expansion of thechannel 20 can provide an accurate means for determining the white celland platelet count when a wide range of variation is expected, as in thecase of abnormally low or abnormally high counts. FIG. 7 shows thelogarithmic slope of the wall of the channel 20 in the insert 16.

FIG. 6 discloses an insert 26 which has a lower end 28 adjacent to or inthe red cell layer and which may be formed with a cylindrical side wallfor a short distance D. The side wall 30 of the insert 26 then slopesupwardly and inwardly toward the axis of the insert 26 at a logrithmeticor other non-linear rate up to the top end 32 of the insert. FIG. 8illustrates the logrithmetic rate of slope of the side wall 30 towardthe axis A. This configuration also permits increased accuracy over awide range by logrithmetically or non-linearly increasing the size ofthe free volume between the insert side wall and the tube bore wall inwhich the buffy layer is disposed.

It has also been found that a distinctly colored material such as dyedparticles of styrene having specific gravities of about 1.035 and 1.075gm/cc may be added to the blood sample and used in conjunction with theaxially elongated insert to sharply define the menisci between theplatelet layer and the white cell lymphocyte layer, and between thelymphocyte layer and the polymorphonuclear-leukocyte (polys) layer. Thisinternal sharp definition within the buffy layer further aids in readingthe white cell and platelet count in accordance with this invention.

FIG. 9 discloses such an embodiment wherein the styrene particles 38 areadhered to the internal bore wall 36 of a capillary tube 34 by means ofan adhesive such as gum acacia soluble in blood and the axiallyelongated insert 35 is likewise adhered to the bore wall 36.

Referring now to FIG. 10, there is shown an insert 40 which is generallycylindrical in shape, but which has at least two adjacent zones withdifferent diameters. The lowermost zone 42 has a larger diameter whichwill provide a greater buffy layer elongation multiple, for examplemultiple of about twenty, and the uppermost zone 44 has a smallerdiameter which will provide a lesser buffy layer elongation multiple,for example a multiple of about three. The two zones 42 and 44 arebridged by a radial shoulder 46. This insert will provide multiplelinear expansion factors for the buffy layer, and can be used to quicklyindicate an abnormally high white cell and platelet count. This highcount will be observed when the buffy layer is expanded above theshoulder 46. The second zone 44 will then give a quick indication of howmuch too high the count is.

Referring to FIG. 11, there is shown an insert 48 which is generallycylindrical in shape and has integral with its upper end 50 an elongatedhandle 52. This handle can aid in packing the inserts in the capillarytubes and will protrude from the ends of the capillary tubes. The handle52 can thus be manually grasped after the blood sample is drawn into thecapillary tube and pumped up and down slowly to cause any dyes in thetube to mix with the blood. The handle 52 can be connected to the insert48 at a weakened area 54 so that the handle 52 can be snapped off of theinsert after mixing but prior to centrifugation.

In order to even further elongate the white cell layer, one couldprovide a coating of microspheres on the external surface of the insertor in any channels formed therein. The microspheres can be adhered toeach other and to the insert by means of a soluble adhesive, such as gumacacia which will dissolve in blood. In the event that some fluid otherthan blood is being tested, the particular adhesive utilized will, ofcourse, be one that is soluble in the fluid being tested.

As previously noted, with the use of an axially elongatedvolume-occupying mass in the tube bore, axial elongation of the layerbeing measured can be increased by a multiple in the range of four toabout twenty. When determining white cell and platelet count in a bloodsample, the preferred range of axial elongation of the white cell layeris a multiple in the range of about five to about fifteen.

It has been discovered that when an axial elongation of the buffy layeris produced within the preferred range, the layering by specific gravityof the individual components of the buffy layer, e.g. thepolymorphonuclears, the mononuclear cells including lymphocytes,monocytes, and platelets. The buffy layer components will layer asfollows in order of lessening specific gravity, the polys, then themonos and lymphocytes (in the same layer), and lastly the platelets.

As previously noted, when the volume-occupying insert is formed from amaterial of the proper specific gravity and has an appropriate axialdimension, the insert will settle slightly into the upper portion of thered cell layer of the centrifuged blood sample. It is in this portion ofthe red cell layer that the reticulocytes, or immature red cells, layerout during centrifugation. Thus the insert will cause axial elongationof the reticulocyte sub-layer of the red cell layer. It has beendiscovered that an approximate reticulocyte count can also be made withthe addition of a fluorescent stain to the blood sample. This count isuseful to the physician in determining the rate of production of new redblood cells by a patient.

To observe this internal layering of the buffy layer, and thereticulocyte layer a fluorescent stain, such as Acridine orange, or thelike, is added to the sample prior to centrifugation. The stain will beabsorbed to varying degrees by the different constituents of the buffylayer, and by the reticulocytes and thus when exposed to fluorescentlight, the various layers will fluoresce to different degrees. Thus thethickness of each sub-layer within the buffy layer and the thickness ofthe reticulocyte layer can be observed by illuminating the tube withlight of the proper wavelength. If desired, optical magnification may beused to observe this layering. The stain may be coated on the tube borewall, may be coated on the insert, or may be inserted into the tube borein the form of a self sustaining, but soluble mass. For use withnon-anticoagulated blood, an anticoagulant such as heparin can be addedin the same manner to the blood sample. It will thus be appreciated thatthis invention makes possible a quick, simple visual diffential whitecell and platelet count and a reticulocyte count through the use of anappropriate fluorescent stain additive and a suitable light source, bothof which may be readily available in a doctor's office.

An apparatus formed in accordance with this invention which will providea nine fold axial expansion of the distance between the upper and lowermenisci of the buffy layer in a sample of centrifuged whole bloodincludes a capillary centrifuge tube having an internal bore diameter of0.05575 in. The volume-occupying mass is a right cylinder made fromRexolite, which is a cross-linked styrene having a specific gravity of1.043 gm/cc, and having a diameter of 0.053 in. and a height of about1/2 in.

It will be appreciated by those skilled in the art that this inventionwill enable visual or mechanical determination of the white cell andplatelet count in a centrifuged sample of whole blood to be made at alow cost and quickly. With proper buffy layer expansion, a differentialwhite cell and platelet determination can be made by using the apparatusand method of this invention. The apparatus of this invention is suchthat it can be prepackaged and can take the form of inexpensivedisposable paraphenalia. Expensive and time consuming cell counters arenot required to practice the technique of this invention.

Since many changes and variations of the disclosed embodiments of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention otherwise than as required by theappended claims.

What is claimed is:
 1. For use in measuring the approximate volume of a constituent material layer in a centrifuged material mixture having at least first and second adjacent material layers of different densities, an apparatus comprising: a tube the bore of which is of substantially constant diameter for containing the centrifuged material mixture; and a volume-occupying mass disposed within the bore in said tube, said mass comprising an axially elongated body formed of a resinous material which sinks through the layer of lesser density but is buoyant upon the layer of greater density, said body having an outside diameter which is sufficiently smaller than the tube bore diameter to form a free space means between said body and said tube bore operable to reduce the available free volume within said tube which substantially all of the layer of lesser density will occupy to axially expand the extent of the layer of lesser density by a multiple of at least about four.
 2. The apparatus of claim 1, wherein said tube is a capillary tube open at both ends for drawing by capillary action a sample of the material mixture.
 3. The apparatus of claim 2, wherein the ends of said capillary tube are inwardly crimped to retain said mass in said tube.
 4. The apparatus of claim 1, wherein said body is a generally cylindrical body having its axis substantially coincidental with the axis of said tube.
 5. The apparatus of claim 4, wherein said cylindrical body includes at least one axially extending groove in its outer side surface which the layer of lesser density occupies.
 6. The apparatus of claim 1, wherein said body has an outer side surface shaped to form means for providing non-linear axial expansion of the layer of lesser density.
 7. The apparatus of claim 1, wherein said body has an outer side surface shaped to form means for providing multiphase linear axial expansion of the layer of lesser density.
 8. The apparatus of claim 1, further comprising colored particulate material of different densities and secured in a cluster to an inner surface of said tube by means of an adhesive material which is soluble in the material being measured, said particulate material being operable to settle into particular interphases between adjacent layers of different density to sharply demarcate the interphases.
 9. For use in measuring the approximate white cell and platelet count in a centrifuged sample of whole anti-coagulated blood, an apparatus comprising: a transparent tube for containing the centrifuged blood sample; at least one volume-occupying body formed from a material having a specific gravity in the range of about 1.02 to about 1.09 gm/cc and disposed in said tube, said body being elongated along an axis which is substantially coincidental with the tube axis, said body being buoyant upon the red cell layer of the centrifuged blood sample, and said body being sufficiently movable within the tube so as to settle onto the red cell layer during centrifugation, said body being radially sized so as to form completely externally of said body a constricted free volume means adjacent to an inner surface of said tube and visible therethrough which constricted free volume means is operative to receive and axially elongate, by a multiple of at least about four, the extent of substantially all of the white cell and platelet layer.
 10. The apparatus of claim 9, wherein said tube is a capillary tube open at both ends for drawing a blood sample.
 11. The apparatus of claim 10, wherein the ends of said capillary tube are radially inwardly crimped to retain said body in said tube.
 12. The apparatus of claim 10, wherein said body is a generally cylindrical body.
 13. The apparatus of claim 12, wherein said cylindrical body has at least one axially extending groove in its side wall forming at least a part of said free volume means.
 14. The appartus of claim 10, wherein said body has a side surface shaped to form a free volume means operative to provide non-linear axial elongation of the extent of the white cell and platelet layer.
 15. The apparatus of claim 10, wherein said body has a side surface shaped to form a free volume means operative to provide multi-phase linear axial elongation of the extent of the white cell and platelet layer.
 16. The apparatus of claim 10, wherein said free volume means is operative to provide axial elongation of the white cell and platelet layer of a multiple in the range of about five to about fifteen and further comprising a stain in said tube which is selectively absorbed by the white cell component cells and the platelets to provide a differential buffy layer count.
 17. The apparatus of claim 16, further comprising colored particulate material of at least two distinctly different densities, said particulate material being adhered together and adhered to a surface within said tube by an adhesive which is soluble in blood, and said particulate material providing means for demarcating at least two different interphases between adjacent sub-component layers in the buffy layer.
 18. For use in measuring the approximate white cell and platelet count in the buffy layer of a centrifuged sample of anticoagulated whole blood, an apparatus comprising a transparent capillary tube for containing the centrifuged blood sample; and volume-occupying means formed from a material having a specific gravity in the range of about 1.02 to about 1.09 gm/cc and disposed in said tube and buoyant upon the red cell constituent of the centrifuged sample for forming a free volume means which is visible through the tube and which is adjacent to the red cell constituent for receiving substantially all of the buffy layer constituent and for axially elongating the distance between the upper and lower menisci of the buffy layer by a multiple of at least abour four.
 19. The apparatus of claim 18, wherein said volume-occupying means is a generally cylindrical body having its axis substantially coincidental with the tube axis.
 20. The apparatus of claim 18 wherein said free volume means is operative to elongate said distance by a multiple in the range of about five to about fifteen.
 21. The apparatus of claim 20, further comprising a stain in said tube which is selectively absorbed to different degrees by the different cell types in the buffy layer to provide for a differential buffy layer count.
 22. The apparatus of claim 21, further comprising colored particulate material of at least two distinctly different densities adhered to a surface within said tube by an adhesive which is soluble in blood, said particulate material providing means for demarcating at least two different interphases between adjacent layers of different cell types in the buffy layer.
 23. For use in measuring the approximate white cell and platelet count in a centrifuged sample of anticoagulated whole blood, an apparatus comprising a capillary tube for containing the centrifuged blood sample; and volume-occupying means formed from a material having a specific gravity in the range of about 1.02 to about 1.09 gm/cc and disposed in said tube and buoyant upon the red cell layer of the centrifuged sample, said volume-occupying means being operable to reduce the free volume within the tube available for containment of substantially all of the white cell and platelet layer by a multiple of at least about 0.75.
 24. For use in measuring the approximate white cell and platelet count in a centrifuged sample of anticoagulated whole blood, an apparatus comprising: a transparent capillary tube for receiving and containing the sample of blood to be centrifuged; a volume-occupying mass disposed within said capillary tube and forming with the bore thereof a free space means which is visible through the tube and is operable to receive and axially elongate substantially all of the white cell and platelet layer of the blood sample by a multiple of at least about four, aid volume-occupying mass being formed of a material which has a specific gravity in the range of about 1.02 to about 1.09 gm/cc and is buoyant upon the red cell layer of the blood sample after centrifugation thereof; and an adhesive material securing said volume-occupying mass to an inner surface of the tube, said adhesive material being soluble in blood.
 25. For use in measuring the approximate differential white cell and platelet count in the buffy layer in a centrifuged sample of anticoagulate whole blood, an apparatus comprising: a capillary tube for drawing and containing the sample of blood to be centrifuged; an axially elongated body disposed in said tube to occupy space therein normally available to the buffy layer, said body being formed of a material which has a specific gravity in the range of about 1.02 to about 1.09 gm/cc and is buoyant upon the red cell layer of the centrifuged blood sample, said body combining with said tube to form a free space means in which substantially all of the buffy layer is disposed with said free space means being operable to elongate the axial extent of the buffy layer by a multiple in the range of about five to about fifteen; and a stain in said tube which stain is absorbed to differing degrees by the several constituent white cell types and the platelets to form within the buffy layer, sub-layers of white cell types and platelets distinguishable by differential coloring.
 26. The apparatus of claim 25, wherein said body includes a handle part protruding from an open end of said tube for manual manipulation in an axial direction to mix the blood and stain prior to centrifugation.
 27. The apparatus of claim 25, further comprising colored particulate material of at least two distinctly different densities adhered to a surface within said tube by an adhesive which is soluble in blood, said particulate material providing means for demarcating at least two different interphases between adjacent sub-layers within the buffy layer.
 28. For use in measuring the approximate reticulocyte count in a centrifuged sample of anticoagulated whole blood, an apparatus comprising: a capillary tube for drawing and containing the blood sample to be centrifuged; an axially elongated body disposed in said tube, said body being formed of a material having a specific gravity in the range of about 1.02 to about 1.09 gm/cc which will cause the body to settle into and float in the red cell layer to an extent sufficient to displace enough of the volume in the tube normally occupied by the reticulocyte layer to form a free space means within the tube operative to receive and axially elongate substantially all of the reticulocyte layer by a multiple in the range of about five to about fifteen; and a stain in said tube which is absorbed by the reticulocyte layer to cause differential coloration thereof. 